Biomedical subjects

X Ye

Publications and source records attributed to X Ye.

At least 127 records · Page 7Linked to original sources

PubMed1999

Central nervous system metabolic and physiologic effects of laparoscopy.

We set out to determine whether the increases in intracranial pressure (ICP) associated with CO2 insufflation had any metabolic effect on the central nervous system in a head injury when compared with gasless laparoscopy (GL). To test this hypothesis, we looked at both the ICP and jugular bulb venous saturation (JVS), with and without a coexisting cerebral mass lesion. Twenty-five kilogram male pigs had tracheostomy, epidural balloon, pulmonary arterial catheter, arterial line, and jugular bulb catheter placed. Intravenous Pentobarbital was used for anesthesia. Either CO2 laparoscopy (CL; n=7) or GL (n=7) were performed both with and without an epidural balloon inflated to a baseline ICP of 25. Data were analyzed using the Student's t test with a P value <0.05 being significant. Cerebral perfusion pressure and most hemodynamic values did not differ. Both central venous pressure and peak inspiratory pressure were significantly elevated whenever CO2 insufflation took place, reflecting an increased intrathoracic pressure. When comparing both study groups, the partial pressure of CO2 did not differ. CL increases ICP significantly above the gasless group in our head injury model. This is most likely secondary to increased intrathoracic pressure. The question still remains whether these changes are clinically significant. We could not demonstrate significant metabolic effects secondary to laparoscopy. In patients suffering head injury, GL rather than CL might be safer to avoid ICP elevation. Additional studies looking at central nervous system metabolic and objective histopathologic effects should be undertaken with larger numbers of study animals.

Animals↗

PubMed1998

Astrocytosis and amyloid deposition in scrapie-infected hamsters.

In scrapie infection, prion protein (PrPSc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrPSc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrPSc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrPSc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrPSc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrPSc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrPSc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrPSc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrPSc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrPSc during some scrapie strain infections. The findings suggest a 'snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.

Amyloidosis↗

PubMed1998

Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse.

The goal of this study is to assess the likelihood that an adenoviral vector disseminated to gonads will be transmitted to offspring. This study is based on the observation that systemically administered vector can be detected in both ovaries and testes, using sensitive nested PCR techniques. Although the extent of vector dissemination to gonads is extremely small, as it is detectable only by nested PCR, it is unclear where it is located within these tissues and whether the DNA is capable of integration and transmission to offspring. A protocol was developed in C3H mice to address this question. Both male and female C3H mice were injected with a high dose of H5.001CBhOTC, an E1- and E4-deleted vector expressing human ornithine transcarbamylase. This dose of vector was sufficient to target 80% of hepatocytes (Gao et al., J. Virol. 1996; 70:8934-8943) and disseminate, at low levels, to both ovaries and testes in 94% of animals as determined by PCR. Vector-administered animals and controls were mated and 814 offspring were evaluated for germ line transmission of the adenoviral vector by DNA hybridization of total cellular DNA extracted from the fetus. Southern blot analysis showed no evidence of germ line transmission in 578 offspring of crosses in which either one or both parents received recombinant adenovirus.

Adenoviridae↗

PubMed1998

Naphthalene-induced oxidative stress and DNA damage in cultured macrophage J774A.1 cells.

Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications including lavatory scent disks, soil fumigants and moth balls. However, little information is available regarding the mechanism of naphthalene toxicity. We have assessed the concentration-dependent in vitro effects of naphthalene on increased lipid peroxidation, cytochrome c reduction, hydroxyl radical production, modulation of intracellular oxidized states by laser scanning confocal microscopy, and DNA fragmentation in cultured macrophage J774A.1 cells. The cells were incubated with 0-500 microM concentrations of naphthalene for 0, 12 and 24 h at 37 degrees C. Concentration- and time-dependent changes were observed. No significant changes were observed with concentrations of naphthalene up to 100 microM. At 24 h, lipid peroxidation increased by 1.8-, 2.4- and 2.9-fold at 200, 300 and 500 microM concentrations of naphthalene. Approximately 2.0-, 3.1- and 4.6-fold increases in cytochrome c reduction were observed at 200, 300 and 500 microM concentrations of naphthalene, respectively, at this time point demonstrating the production of superoxide anion, while under the same conditions approximately 2.4-, 3.2- and 4.9-fold increases in hydroxyl radical production were observed, respectively. Following incubation of these cells with 200 and 500 microM concentrations of naphthalene 2.3- and 4.7-fold increases in fluorescence intensity were observed, respectively, as compared to the untreated cells. At 24 h, approximately 1.8-, 2.3- and 3.0-fold increases in DNA fragmentation were observed following incubation with 200, 300 and 500 microM concentrations of naphthalene, respectively. Naphthalene also produced concentration- dependent decreases in cell viability. At the 12 h time point, significant changes were observed only with 300 and 500 microM concentrations of naphthalene. These results demonstrate that naphthalene may induce toxic manifestations by enhanced production of oxygen free radicals, resulting in lipid peroxidation and DNA damage.

Animals↗

PubMed1998

Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription.

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

Animals↗

PubMed1998

Activation mechanism of anticoagulant protein C in large blood vessels involving the endothelial cell protein C receptor.

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin-TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell-specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin-TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.

Antibodies, Monoclonal↗

PubMed1998

Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses.

Preclinical studies were designed to investigate the feasibility and safety of recombinant adenoviruses transduced into the hepatic artery of nonhuman primates. The vectors used are recombinant adenoviruses deleted in E1 and contain either a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein at nonpermissive temperatures, or a deletion of the E4 region, including open reading frame (ORF) 6. Six 8- to 10-kg baboons underwent femoral artery cannulation, and angiographic techniques were used to introduce vector selectively into either a portion of the right lobe of the liver via a branch of the right hepatic artery or the common hepatic artery. Necropsies were performed at 4, 29, or 61 days. Serial sequential liver biopsies were performed in the baboons that survived 29 or 61 days. In the 2 baboons with vector transduction into the right hepatic artery, X-Gal histochemical analysis of the liver showed evidence of quantitatively increased gene transfer in the targeted lobe; however, gene transfer was present throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was present throughout both livers transduced with the highest dose of vector. No differences were seen in the level of portal inflammation in targeted and untargeted lobes despite the observed qualitative and quantitative differences in gene expression. Southern blot analysis of total cellular DNA isolated from targeted and nontargeted lobes showed similar levels of viral DNA throughout the liver. Polymerase chain reaction (PCR) analysis was able to detect viral DNA sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector. In baboons treated with lower doses of an E1-E4 deleted vector expressing the human ornithine transcarbamylase (OTC) gene, DNA was detectable by nested PCR in liver but not gonads at days 29 and 61. The data suggest that intraarterial administration of recombinant adenoviral E1-E4 deleted vector is feasible and safe. At high doses of vector, widespread dissemination of vector DNA is seen. At low doses, hepatic gene transfer is not associated with vector DNA dissemination to gonads.

Adenoviridae↗

PubMed1998

Tools for visualization and integration of intermediate sequencing results in large disease gene discovery projects.

We describe two Java applets which are useful for insightful presentation of intermediate experimental data in gene discovery projects involving large scale sequencing. One of these applets provides a physical map of a genomic region and provides easy access to the second applet, which furnishes a detailed map of sequence contigs associated with clones on the physical map. In particular, the second applet displays all the known information about each contig, including the presence of exons, database homology 'hits', repetitive elements and other features; the graphics are linked to other World Wide Web pages, providing detailed information on each feature. These applets should be useful to other research groups working on large sequencing projects.

Chromosome Mapping↗

PubMed1998

High-resolution YAC-cosmid-STS map of human chromosome 13.

We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.

Animals↗

PubMed1998

Protein phosphorylation associated with epipodophyllotoxin-induced apoptosis of lymphoid cells: role of a serine/threonine protein kinase.

We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca2+ was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca2+-independent. In the present study, we demonstrate that addition of 10 microM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 microM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number of de novo phosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests that de novo protein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases.

Animals↗

PubMed1998

In vivo measurement of ureagenesis with stable isotopes.

We have utilized stable isotopes to measure in vivo rates of ureagenesis. In one testing procedure, 15NH4Cl was administered orally to controls and to heterozygotes for ornithine transcarbamylase deficiency. Controls produced [15N]urea at a rate that was greater than that of symptomatic carriers, but indistinguishable from that of asymptomatic carriers. In contrast, both symptomatic and asymptomatic heterozygotes produced [5-15N]glutamine more rapidly than the controls. Ureagenesis could also be measured by administering sodium [1-13C]acetate to a healthy adult and measuring subsequent formation of [13C]urea. The latter approach involves the use of isotope ratio mass spectrometry to determine isotopic abundance. This technique is much more sensitive than gas chromatography-mass spectrometry for the measurement of isotopic label, a consideration that makes the method more suitable for the study of subjects in whom ureagenesis is severely compromised, for example the human male neonate with a near complete deficiency of ornithine transcarbamylase.

Adult↗

PubMed1998

Developing adenoviral-mediated in vivo gene therapy for ornithine transcarbamylase deficiency.

There are a number of reasons for choosing ornithine transcarbamylase (OTC) deficiency as a candidate for gene therapy: the gene has been cloned; the disorder is relatively common; the current clinical outcome is poor; and there are authentic animal models. In considering the development of gene therapy for OTC deficiency, we focused on the use of in vivo gene therapy with an adenoviral vector. Using the partially OTC-deficient sparse fur mouse we found transduction and expression could be achieved using an intravenous infusion of a recombinant adenovirus containing the OTC cDNA. The results were transient as a result of immune activation in response to the vector and vector-transduced cells. By modifying the adenoviral construct, creating an E1 deletion-E2 temperature-sensitive mutation, we blunted the cytotoxic T lymphocyte immune response and achieved correction of biochemical abnormalities for 2-3 months. We also found that transduction and expression following gene transfer occurred sufficiently rapidly to protect against acute hyperammonaemia within 24 h. Subsequent preclinical studies in mice and non-human primates demonstrated that E1-E4-deleted vectors had a substantially improved safety profile and similar efficacy. With this evidence of efficacy and safety of adenoviral vectors, we are embarking on a phase I trial of intravascular gene transfer using an E1-E4-deleted vector in adults with partial OTC deficiency.

Adenoviruses, Human↗

PubMed1998

3D HCCH-COSY-TOCSY experiment for the assignment of ribose and amino acid side chains in 13C labeled RNA and protein.

A new 3D HCCH-COSY-TOCSY experiment is presented for the assignment of RNA sugar and protein side chains. The experiment, which combines COSY and TOCSY units, is more powerful than the sum of individual HCCH-COSY and HCCH-TOCSY pulse sequences. The experiment was applied to a 13C, 15N-labeled 26 mer RNA complexed with the antibiotic tobramycin, and a 12 kDa 13C, 15N-labeled FKBP12 protein sample. The power of HCCH-COSY-TOCSY is demonstrated through complete spin system assignments of sugars in the 26 mer RNA sample, which could not be assigned using a combination of HCCH-COSY, HCCH-TOCSY and 13C-edited NOESY experiments.

Amino Acids↗

PubMed1998

Solution structure of P22 transcriptional antitermination N peptide-boxB RNA complex.

We have determined the solution structure of a 15-mer boxB RNA hairpin complexed with a 20-mer basic peptide of the N protein involved in bacteriophage P22 transcriptional antitermination. Complex formation involves adaptive binding with the N peptide adopting a bent alpha-helical conformation that packs tightly through hydrophobic and electrostatic interactions against the major groove face of the boxB RNA hairpin, orienting the open opposite face for potential interactions with host factors and/or RNA polymerase. Four nucleotides in the boxB RNA hairpin pentaloop form a stable GNRA like tetraloop structural scaffold on complex formation, allowing the looped out fifth nucleotide to make extensive hydrophobic contacts with the bound peptide. The guanidinium group of a key arginine is hydrogen-bonded to the guanine in a loop-closing sheared G.A mismatch and to adjacent backbone phosphates. The identified intermolecular contacts account for the consequences of N peptide and boxB RNA mutations on bacteriophage transcriptional antitermination.

Amino Acid Sequence↗

PubMed1998

p75NTR and the concept of cellular dependence: seeing how the other half die.

Cells depend on specific stimuli, such as trophic factors, for survival and in the absence of such stimuli, undergo apoptosis. How do cells initiate apoptosis in response to the withdrawal of trophic factors or other dependent stimuli? Recent studies of apoptosis induction by neurotrophin withdrawal argue for a novel form of pro-apoptotic signal transduction - 'negative signal transduction' - in which the absence of ligand-receptor interaction induces cell death. We have found that the prototype for this form of signaling - the common neurotrophin receptor, p75NTR - creates a state of cellular dependence (or addiction) on neurotrophins, and that this effect requires an 'addiction/dependence domain' (ADD) in the intracytoplasmic region of p75NTR. We have recently found other receptors that include dependence domains, arguing that dependence receptors, and their associated dependence domains, may be involved in a rather general mechanism to create cellular states of dependence on trophic factors, cytokines, adhesion, electrical activity and other dependent stimuli.

Animals↗

PubMed1998

Measurement of genetic variation within and between Japanese quail lines using DNA fingerprinting.

The objective of the present experiment was to study genetic variation within and among well-defined Japanese quail lines by DNA fingerprinting. The Japanese quail lines included a randombred control line (R1) and lines developed from R1 by divergent selection over 30 generations for 4-wk BW (HW, LW) and total plasma phosphorus (TPP) (HP, LP), a measure of yolk precursor in the blood. In addition, two sublines (HW-HP, HW-LP) of HW, developed in the ninth generation, were included in the analysis. Males of the sublines were selected for increased 4-wk BW whereas females were selected for increased (HW-HP) or decreased (HW-LP) TPP. Sixteen individual DNA samples per line were digested with HaeIII restriction enzyme and hybridized with Jeffreys' 33.6 probe. The DNA fingerprints were analyzed with computer programs designed to measure band sharing (BS). Within lines, BS ranged from 0.384 to 0.525. The BS within the R1 line was less than that of all selected lines, except for the HP and LP lines, indicating that, in general, selection had increased genetic homogeneity within the selected lines. Between lines, BS was less than within lines and the R1 line had the highest average level of BS (0.278) with the other lines. The BS between lines for the selected lines ranged from 0.230 to 0.308 with an average of 0.265. In the comparison of the R1 line with the selected lines, it appeared that selection for increased TPP or decreased BW may have influenced BS levels. The relationships of the HW line with its sublines (HW-HP and HW-LP) were not accurately predicted by the DNA fingerprinting technique used. All lines were separated, as indicated by the genetic distance between lines.

Animals↗

PubMed1998

Genetic diversity of commercial turkey primary breeding lines as estimated by DNA fingerprinting.

The genetic diversity of primary breeding sire and dam lines from the three largest turkey breeders was estimated by band sharing of DNA fingerprints and by genetic distance estimated from band sharing. For comparison, experimental lines selected for increased egg production (E line) or increased 16-wk body weight (F line) were also included in the analysis. Eighteen individual DNA samples per line were digested with HaeIII restriction enzyme and hybridized with Jeffreys' 33.6 probe. The DNA fingerprints were analyzed with computer programs designed to measure band sharing. Within commercial lines, band sharing ranged from 0.370 to 0.508 and was greater in commercial sire lines (average = 0.475) than in commercial dam lines (average = 0.393), indicating that accumulated inbreeding was greater in the sire lines. Band sharing in the F and E lines was 0.479 and 0.522, respectively. The average band sharing among lines was higher for primary breeding sire lines (average = 0.267) than for primary breeding dam lines (0.207), suggesting more genetic diversity in the dam lines. Genetic distance estimated from band sharing was greater among commercial dam lines than commercial sire lines. Based on band sharing between lines and genetic distance estimates, it appears that the experimental E and F lines contain genetic variation not found in the commercial lines. The results of the present study, along with data published in the literature, suggest that commercial primary breeding turkey lines are as diverse, if not more diverse, than similar commercial chicken lines.

Animals↗

PubMed1998

Regulation of lung fibroblast tropoelastin expression by alveolar epithelial cells.

Epithelial-mesenchymal interactions are of critical importance during tissue morphogenesis and repair. Although the cellular and molecular aspects of many of these interactions are beginning to be understood, the ability of epithelial cells to regulate fibroblast interstitial matrix production has not been extensively studied. We report here that cultured alveolar epithelial cells are capable of modulating the expression of tropoelastin, the soluble precursor of the interstitial lung matrix component elastin, by lung fibroblasts. Phorbol ester-stimulated alveolar epithelial cells secrete a soluble factor that causes a time- and dose-dependent repression of lung fibroblast tropoelastin mRNA expression. This alveolar epithelial cell-mediated repressive activity is specific for tropoelastin, is effective on lung fibroblasts from multiple stages of development, and acts at the level of transcription. Partial characterization of the repressive activity indicates it is an acid-stable, pepsin-labile protein. Gel fractionation of alveolar epithelial cell conditioned medium revealed two peaks of activity with relative molecular masses of approximately 25 and 50 kDa. These data support a role for epithelial cells in the regulation of fibroblast interstitial matrix production.

Animals↗